1Department of Biology, University of Puerto Rico, Río Piedras, San Juan, Puerto Rico.

Abstract

Metazoan immunity is mainly associated with specialized cells that are directly involved with the immune response. Nevertheless, both in vertebrates and invertebrates other organs might respond to immune activation and participate either directly or indirectly in the ongoing immune process. However, most of what is known about invertebrate immunity has been restricted to immune effector cells and little information is available on the immune responses of other tissues or organs. We now focus on the immune reactions of the intestinal tissue of an echinoderm. Our study employs a non-conventional model, the echinoderm Holothuria glaberrima, to identify intestinal molecules expressed after an immune challenge presented by an intra-coelomic injection of lipopolysaccharides (LPS). The expression profiles of intestinal genes expressed differentially between LPS-injected animals and control sea water-injected animals were determined using a custom-made Agilent microarray with 7209 sea cucumber intestinal ESTs. Fifty (50) unique sequences were found to be differentially expressed in the intestine of LPS-treated sea cucumbers. Seven (7) of these sequences represented homologues of known proteins, while the remaining (43) had no significant similarity with any protein, EST or RNA database. The known sequences corresponded to cytoskeletal proteins (Actin and alpha-actinin), metabolic enzymes (GAPDH, Ahcy and Gnmt), metal ion transport/metabolism (major yolk protein) and defense/recognition (fibrinogen-like protein). The expression pattern of 11 genes was validated using semi-quantitative RT-PCR. Nine of these corroborated the microarray results and the remaining two showed a similar trend but without statistical significance. Our results show some of the molecular events by which the holothurian intestine responds to an immune challenge and provide important information to the study of the evolution of the immune response.

Vertical lines indicate the two-fold change threshold, up (>1) or downregulation (<−1), to determine differentially expressed genes. Points below the horizontal line are not statistically significant. Each dot represents a single probe on the microarray. Dots located in the upper-left and upper-right cuadrants represent differentially expressed probes (down-regulated and up-regulated, respectively) at P<0.01.

For each sequence a gel image shows the PCR amplification of the gene and the control NADH (second lower band), except for Hg_Act1, where the actin band is lower than that of NADH. Each lane represents RT-PCR products from RNA pooled from three different animals. Graph bars indicate the averaged OD ratios between each gene and NADH for three different experiments (each with a different pool of animals). Lines represent standard deviation. Asterisks represent t-test significance (*P<0.05, **P<0.01).